Metastasis can occur through two routes, hematogenous or lymphatic. The hematogenous route results in distant metastases, whereas lymphatic metastases tend to colonize nodes following the route of the draining lymphatics. Lymph node metastases are more frequent than hematogenous metastases in cancer overall, yet this form of metastasis has been tittte studied in the laboratory. Part of the difficulty in approaching lymphatic metastasis have been the limitations of the current animal models available for modeling lymph node metastasis. Here we propose to develop methods to detect tumor cell spread to the lymph nodes in murine model systems. These systems wilt then be used to test and refine novel methods for the detection of lymph node metastases. In our preliminary data we have used subcutaneous tumor formation in the mouse thigh leading to the colonization of the draining lymph nodes to recapitulate lymphatic metastasis. From tumor formation at that site tumor ceils can gain access to the femoral and inguinal lymphatics and travel beyond to the para-aortic chain. We now propose to develop these observations into quantitative assays to allow the study of lymphatic metastasis in live animals. We plan to use a variety of methods to produce a quantitative assessment of the number of tumor cells in the lymph node. A reliable and sensitive determination of lymph node metastasis will then allow application of this assay to the development of optical methods to detect lymph node metastasis in patients. Currently the detection of lymph node metastasis is crucial for clinical diagnosis. In most cases the presence of lymph node metastasis predicts a worse prognosis. Because of the critical predictive value of lymph node metastasis in the clinical staging of cancer, strategies have been developed to enhance the ability of clinicians to find involved lymph nodes. Since lymph node metastases tend to track along the lymphatics draining the tumors, evaluation of nodes along those lymphatics has proven to be particularly valuable and these nodes are called sentinel nodes for that reason. A reliable assay of lymph node metastasis will be invaluable for developing new methods for detection of tumor metastatic to lymph nodes and in refining the current procedures for sentinel node biopsy, it should also help in determination of the minimum number of cells that can be detected in lymph nodes using various strategies. Additionally a reliable assay will further the study of the mechanisms responsible for tymph node metastasis and to assess therapeutic modalities specifically designed to attack lymph node metastases.